Di- and trihydroxy aromatics are important industrial chemicals with many applications as evidence by worldwide production of catechol, resorcinol, and hydroquinone at 110,000 tons/year. Catechol is used as an intermediate in the food, pharmaceutical, and agrochemical industries, and hydroquinone is used in photography, in cosmetics, and in both medical and industrial X-ray films. Substituted catechols, especially 3-substituted catechols, are useful precursors for making pharmaceuticals; one of these, 3-methoxycatechol is an important intermediate for the antivascular agents combretastatin A-1 and combretastatin B-1. Hydroxyquinone and its derivatives are important chemicals used mainly as photographic developers, polymerization inhibitors, rubber antioxidants, food antioxidants, synthesis intermediates, and also used in water treatment. Methoxyhydroquinone is used in the synthesis of triptycene quinones that have been shown to have anti-leukemia cell activity. Resorcinol and its derivatives are used to inhibit rust in paints, to regulate plant growth, and to act as capacitor electrolytes. Production of 4-methylresorcinol is uncommon and prices can exceed $200,000/kg (Apin Chemicals). Methylhydroquinone has been recently reported to be used in the synthesis of (±)-helibisabonol A, and puraquinonic acid which are precursors to agrochemical herbicides and antileukemia drugs, respectively. 1,2,3-Trihydroxybenzene (1,2,3-THB, pyrogallol), the first synthetic dye for hair, is primarily used as a modifier in oxidation dyes, as a pharmaceutical intermediate, and has been used as a topical antipsoriatic. Hydroxyhydroquinone (1,2,4-THB) has been used in dyes and as a corrosion inhibitor. Manufacture of these substituted dihydroxylated compounds by chemical routes is difficult due to the employment of aggressive reagents, expensive and complicated starting materials, multiple reaction steps, and low yields. Direct microbial oxidation of NB or NPs for the synthesis of NC or NHQ is attractive to reduce wastes (relative to organic-based methods) since chemical synthesis of these compounds is problematic in terms of yield and selectivity.
Nitroaromatic compounds are widely used in industry as dyes, pesticides, plasticizers, explosives, and solvents, and dihydroxy nitroaromatics are important for medicine. Nitrocatechol derivatives have been shown to be selective and potent inhibitors of catechol-o-methyltransferase, which is important in the metabolism of catechol drugs, and so nitrocatechol derivatives may be used in the treatment of Parkinson disease. Nitrocatechols have been found to be useful intermediates for the synthesis of pharmaceuticals such as Flexinoxan, an antihypertensive drug. 4-Nitrocatehcol (4-NC) and 3-NC have potential for therapeutic interest, and were recently found to be competitive inhibitors of nitric oxide synthase with potential anti-nociceptive (pain relieving) activity. 3-NC is also essential as a building block for the production of some antihypertensive pharmaceutical such as flesinoxan. Nitrohydroquinone (NHQ) has been used to synthesize dephostatin; an inhibitor of the protein tyrosine phosphatase with is a candidate therapeutic agent for diabetes mellitus and neural diseases such as Alzheimer's disease and Parkinson's disease. Industrially, 3-NC is also useful for electrolytic capacitors operating at high temperatures or used to increase the amplification factor of transistors. NHQ is mainly used as electrophotographic photoreceptor, and dyes.
As chemical synthesis of these compounds is problematic in terms of yield and selectivity, the utilization of oxygenases is advantageous. The high redox potential of oxygenases enables them to perform reactions with chemically stable substrates as well as provide a high degree of region and enantioselectivity. Transforming selectively an inexpensive and abundant chemical as nitrobenzene (NB) into a valuable feedstock for drug production, namely 4-NC, is therefore of great significance.
There have been previous reports in the literature on oxygenases capable of producing nitrocatechols. p-Nitrophenol hydroxylase of Arthrobacter sp. and Bacillus sphaericus JS905 transforms p-nitrophenol (p-NP) to 4-NC often with further removal of the nitro group to obtain 1,2,4-trihydroxybenzene (Jain et al., 1994; Kadiyala and Spain, 1998). Kieboom and co-workers screened twenty-one microorganisms for their ability to convert nitroaromatics into 3-NC. Strains containing toluene-dioxygenases from P. putida F1, Nocardia S3, Pseudomonas JS150, Cornybacterium C125, and Zanthobacter 124X were able to transform NB to 3-NC rapidly. They did not report a toluene monooxygenase-containing strain able to perform this reaction. Haigler and Spain reported Pseudomonas mendocina KR1 and Ralstonia pickettii PKO1 convert NB to NC; however, the enzymes responsible for the addition of the second hydroxyl group to the nitrophenols to form nitrocatechols were not identified. Pseudomonas mendocina KR-1 converts NB to 4-NC via m-NP (10%) and p-NP (63%), and Pseudomonas pickettii PKO1 converts NB to 3-NC and 4-NC via m-NP and p-NO. Pseudomonas putida 2NP8 grown on m-NP has been shown to degrade NB into ammonia, nitrobenzene, and hydroxylaminobenzene. O—NP is degraded by this strain with production of nitrite, and m-NP resulted in the formation of ammonia. Pseudomonas pseudoalcoligenes JS45 degrades NB to 2-aminomuconate, which is also an intermediate in the metabolism of tryptophan in mammals.
Twenty-one oxygenase-containing bacteria were screened for the ability to convert nitroaromatics into 3-NC. Mycobacterium chelonae strain NB01 was shown to degrade NB via reductive degradation mechanism, which resulted with the formation of ammonia. Comamonas sp strain JS765 was shown to convert NB to an unstable nitrohydrodiol that spontaneously decomposes to form catechol and nitrite via nitrobenzene 1,2-dioxygenase.
Indigo is one of the oldest dyes and is still used worldwide for textiles with 22,000 tons produced annually worth $200 million. Historically, this blue dye was obtained from various plant sources, including woad (Isatis tinctoria) in Europe and Indigofera in Asia and South America. Now production of indigo is primarily by the Adolf von Baeyer 1890 chemical synthesis which resulted in the fifth Noble Prize in chemistry. More recently, bacterial systems for commercial indigo production have been developed, which were inspired by the discovery that growth of the recombinant Escherichia coli strain expressing naphthalene dioxygenase from Pseudomonas putida PpG7 in rich medium resulted in the formation of indigo. Indigo is formed and the result of the cloned enzyme oxygenating C-3 of the indole pyrrole ring, and indole is produced from tryptophan via tryptophanase in E. coli. Various monooxygenases and dioxygenases have been identified that are capable of indole oxidation to form indigo, and these biological processes are inherently safer than the Adolf von Baeyer process since they do not produce such toxins as aromatic amines (bladder carcinogens), and cyanide.
Indirubin, a pink pigment, is also produced in minor amounts from plant sources. Due to the small and variable amount of indirubin, plant-derived indigo dye has a more pleasing tinge than synthetic indigo. In addition, indirubin has important and potential therapeutic applications since it is the active ingredient of a traditional Chinese medicine used to treat diseases such as chronic myelocytic leukemia (CML) and was found to be a potent inhibitor of cyclin-dependent kinases and therefore belongs to a group of promising anticancer compounds.
Some of these compounds cannot be easily synthesized chemically, and the traditional chemical processes are often lengthy and require expensive starting materials. Direct microbial synthesis of such compounds from inexpensive substrates might provide a more cost effective and more environmentally benign approach, and biocatalysis is likely to account for 30% of the chemical business by 2050. Biocatalysis has become an attractive alternative to chemical synthesis because of its high selectivity and efficiency. Since 2000, more than 400 patents on the use of microorganisms or enzymes to produce specialty chemical shave been issued. Among the various classes of enzymes, oxygenases are considered one of the most promising due to their ability to perform selective hydroxylation that are not accessible by chemical methods. One recent commercial example is the production of an intermediate for an antilipolytic drug from the oxidation of 2,5-dimethylpyrazine to 5-methylpyrazine-2-carboxylic acid with whole cells of Pseudomonas putida ATCC 33015 expressing xylene monooxygenase. For example, it can produce relatively pure compounds compared with racemic mixtures often obtained by chemical methods. Biocatalysis also avoids tedious blocking and deblocking steps, which are common in the chemical synthesis of enantio- and regioselective compounds, and is inherently environmentally benign as the reactions are usually performed in water (avoiding harsh solvents) at room temperature and atmospheric pressure under milder conditions.
More recently, a large number of enzymes have been studied for aromatic hydroxylations such as heme P450s, flavin monooxygenases, pterin-dependent non-heme monooxygenases, non-heme mononuclear iron dioxygenases, and diiron hydroxylases. For example, Meyer et al. (2002) reported that directed evolution using error-prone PCR increased the substrate specific activity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase 2 times towards o-methoxyphenol and 5 times towards 2-tert-butylphenol for making the corresponding 3-substituted catechols. Canada et al. (2002) used DNA shuffling to evolve toluene ortho-monooxygenase (TOM) from Burkholderia cepacia G4 for 1-naphthol synthesis, and one mutant (TomA3 V106A) with 6-fold increased activity was found. Furthermore, substituted catechols (e.g., 3-bromocatechol, 3-methoxycatechol, 3-iodocatechol, 3-methylcatechol) were synthesized from substituted benzenes in two steps using recombinant E. coli expressing both toluene dioxygenase and dihydrocatechol dehydrogenase.
Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1 belongs to the family of diiron hydroxylases including the methane, toluene, benzene, o-xylene monooxygenases, phenol hydroxylases, and alkene epoxidases. T4MO is a soluble, non-heme, O2-dependent, diiron monooxygenase, and is a four-component alkene/aromatic monooxygenase enzyme consisting of six genes designated tmoABCDEF. The genes tmoA, tmoB, and tmoE encode the •, •, and • subunits, respectively. The hydryolase component (212-kDa with (•••)2 quaternary structure) which was recently described as responsible for the regiospecificity of the enzyme. Gene tmoF encodes a 36-kDa NADH oxidoreductase containing FAD and a [2Fe-2S] cluster. The tmoC encodes a 12.5-kDa Rieske-type [2Fe-2S] ferredoxin involved in electron transfer between the hydroxylase and reductase; tmoD gene encodes an 11.6-kDa catalytic effector protein. All four protein components from the 6 genes are required for efficient multiple catalysis and high regiospecificity. The (•••)2 hydroxylase component containing the active site for substrate binding and hydroxylation reaction (Pikus et al., 1997) was reported recently to be responsible for the monooxygenation regiospecificity of T4MO while the binding of the effector protein refined the product distribution leading to high regiospecificity. The binding effector protein has been shown to enhance the catalytic rate of the enzyme and to refine the product distribution leading to the high regiospecificity of T4MO.
T4MO is a highly regiospecific enzyme, hydroxylating nearly all monosubstituted benzenes tested including toluene, chlorobenzene, methoxybenzene, and nitrobenzene at the para position. Recent mechanistic studies reveal that active site-directed opening of an epoxide intermediate may account for this high regiospecificity. T4MO has been shown to perform single hydroxylations, transforming benzene to phenol, toluene to p-cresol and other monosubstituted benzenes to the subsequent p-hydroxylated compounds. Wood and co-workers have recently reported that T4MO expressed in Escherichia coli TG1 cells can perform successive hydroxylation, resulting in conversion of benzene to 1,2,3-trihydroxybenzene. Nevertheless, there is no evidence to date of T4MO being able to convert substituted benzenes (e.g., nitrobenzene) to their respective catechols (e.g., nitrocatechol). T4MO is the most efficient enzyme towards toluene oxidation among toluene monooxygenase family including TOM, toluene para-monooxygenase (formerly toluene 3-monooxygenase) of Ralstonia picketti PKO1, and toluene/o-xylene monooxygenase of Pseudomonas stutzeri OX1. T4MO has been identified to oxidize toluene to 96% p-cresol, 3% m-cresol, and less than 1% benzyl alcohol. Other enzymes, for example, ammonia monooxygenase, chloroperoxidase, cytochrome P450, methane monooxygenase, and xylene monooxygenase oxidize alkylbenzenes; however, they produce benzyl alcohols (70-100% of total products) and only negligible amounts of phenolic products. The high regiospecificity for para hydroxylation of toluene and nearly no ortho activity make T4MO a valuable and rare enzyme that is specialized for aromatic ring hydroxylation. In addition, T4MO has broad substrate specificity for mono-substituted benzenes including nitrobenzene, chlorobenzene, and methoxybenzene, which are catalyzed to single hydroxylated products in the para position.
Toluene-o-Xylene Monooxygenase (ToMO) hydroxylates toluene in the ortho, meta, and para positions as well as o-xylene in both the 3 and 4 positions, and it oxidizes many substrates including o-xylene, m-xylene, p-xylene, toluene, a benzene, ethyl-benzene, styrene, naphthalene, and trichloroethylene (TCE), and is the only known oxygenase which attacks tetrachloroethylene. The six genes coding for ToMO are touABE (three-component hydroxylase with two catalytic oxygen-bridged dinuclear centers, A2B2E2), touC (ferredoxin), touD (mediating protein), and touF (NADH-ferredoxin oxidoreductase). ToMO touA (499 amino acids has the greatest amino acid identity to the hydroxylase (TbuA1) of toluene 3-monooxygenase (T3MO) of Pseudomonas picketti PKO1 (68%) and the hydroxylase (TmoA) of toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 (66.8%), but these are distinct enzymes given their different regiospecific oxidation of toluene.
The importance of position V106 as an active residue in toluene monooxygenases was reported previously by us as a result of directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4. This beneficial mutation resulted in a two-fold increase in the initial degradation rate for TCE degradation and a six-fold increase for naphthalene oxidation. This position corresponds to I100 of the alpha subunit TouA of the hydroxylase in ToMO.
The methane monooxygenase (MMO) active site residues have been identified by X-ray crystallography, and by comparison to MMO, some of these active site residues for T4MO, T3MO, and toluene 2-monooxygenase from Pseudomonas sp. strain JS150 have been predicted by Pikus et al. (1997); hence, several positions in the alpha subunits of aromatic monooxygenases have been studied. Position T201 of tmoA of T4MO, and positions T201, Q141, and F205 of TouA of ToMO (Vardar and Wood, 2004) have been studied via saturation mutagenesis. T4MO mutants Q141C, Q141V, I180F, and F2051 of tmoA have been studied previously via site directed mutagenesis; the same residues (except M180) and positions are the same for ToMO. For T4MO TmoA mutant Q141C, oxidation of m-xylene to 3-methylbenzyl alcohol formation increased 6-fold from 2.2% to 1.7%, and for p-xylene oxidation, the product distribution completely switched to 2,5-dimethylphenol (78%) from 4-methylbenzyl alcohol (22%). T4MO tmoA mutant T201F gave a large shift in the product distribution and also formed 10-fold more benzyl alcohol from toluene. For the hydroxylation of toluene by T4MO mutant F2051 of tmoA, the percentage of m-cresol formation increased 5-fold from 2.8% to 14.5%. The TouA F205G mutation in ToMO changed the hydroxylation regiospecificities of toluene, o-cresol, m-cresol, p-cresol, phenol, and resorcinol, and allowed for the novel formation of methylhydroquinone, 4-methylresorcinol, hydroquinone, resorcinol, and 1,2,3-trihydroxybenzene (Vardar and Wood, 2004). T4MO mutants from positions Q141, T201, and F205 were not studied previously for nitrobenzene oxidation with the exception of T4MO mutant T201G of TmoA that produced 7.9% o-NP whereas wild-type T4MO did not.
Burkholderia capacia G4 was isolated as the first pure strain that degrades trichloroethylene (TCE), and toluene ortho-monooxygensase (TOM) has been shown to oxidize mixtures of cholorinated compounds, including TCE (Shim and Wood, 2000). The subunit of TOMs are similar to the corresponding components of crystallographically-characterized soluble methane monooxygenase (sMMO) from methanotrophic bacteria, with their •-subunits sharing about 20% amino acid sequence identity. TOM is a three-component complex consisting of a 211-kDa hydroxylase (tomA1A3A4), with two binuclear iron centers in the (•••)2 quaternary structure, a 40-kDa NADH-oxidoreductase (tomA5), and a 10.4-kDa cofactor-less regulatory protein (tomA2) involved in the electron transfer between the hydroxylase and reductase. The (•••)2 component contains the active site for substrate binding and hydroxylation reaction and is capable of a peroxide-shunt mechanism like sMMO.
TOM originally was not considered as an indigo-forming strain, but our laboratory found it was responsible for color development and indole hydroxylation. During growth in complex medium, recombinant E. coli expressing TOM forms brown color on agar plates an in liquid culture, whereas typical indole-oxygenating enzymes in whole cells from blue colonies on agar plates and blue, water-insoluble pigments in liquid medium. In addition, one TOM variant created from DNA shuffling was identified as a potential indigo-forming enzyme; based on the color of its colonies on agar plates and in liquid culture it was termed TOM-Green with a single amino acid change of valine to alanine at position V106 of the hydroxylase a-subunit (TomA3). Thus in this variant, a single mutation was responsible of the cell color change, presumably due to the alteration in the hydroxylation of indole.
DNA shuffling is a widely used method for protein mutagenesis in which there is no need for crystal structure or any information about the structure of the protein. Using DNA shuffling, the TomA1 V106A mutation of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 was identified (corresponds to I100 of the alpha subunit TouA of the hydroxylase in ToMO) which resulted in a 2-fold increase in the initial degradation rate for TCE degradation and a 6-fold increase for naphthalene oxidation. The importance of position I100 was corroborated in saturation mutagenesis for TmoA of T4MO and TouA of ToMO (Vardar and Wood, 2004). T4MO TmoA mutant I100L was found to have a 4-fold increase in activity for 3-methoxycatechol formation from 1 mM guaiacol. In addition, T4MOTmoA mutant I100A and I100S produced 20% m-cresol and 80% p-cresol, whereas the wild-type T4MO produced 96% p-cresol. ToMO TouA variant I100Q had significantly altered hydroxylation regiospecificities for toluene, o-cresol, m-cresol, phenol, and catechol allowing for the novel formation of methylhydroquinone, hydroquinone, and 1,2,4-trihydroxybenzene (Vardar and Wood, 2004).
Despite efforts to date, a need remains for improved system(s), method(s) and/or process(es) for generating desired monooxygenase enzymes and related polypeptides.